Inositol is found in nature in a large number of plants. In yeast, and possibly higher animals, inositol appears to play an essential role in membrane phospholipid balance and is required for proper growth. Greenberg et al., Genetics, 100:19-33 (January 1982). The standard commercial source for inositol is corn steep liquor, since inositol is present as phytic acid in corn. See Artz et al., U.S. Pat. No. 2,615,053.
Greenberg et al. reported on regulatory mutations of inositol biosynthesis in yeast. They isolated several inositol excreting mutants of Saccharomyces cerevisiae. In this microorganism the enzyme inositol-1-phosphate synthase (I-1-P synthase), product of the INO1 gene locus, catalyzes the first step in inositol biosynthesis, i.e., the synthesis of inositol-1-phosphate from the substrate glucose-6-phosphate. In the wild type strain the activity of this enzyme is dramatically repressed in the presence of inositol. By selecting for mutants which overproduce and excrete inositol, Greenberg and coworkers idnetified mutants constitutive for inositol-1-phosphate synthase. Genetic analysis of the mutants indicated that at least three loci, designated OPI1, OPI2 and OPI4, direct inositol-mediated repression of I-1-P synthase. Mutants of these loci synthesize I-1-P synthase constitutively. Inositol excretion in the mutant strains was identified by replicating a colony of one of the excretory mutants onto selection plates followed by incubation for 5-10 days at 30.degree. C. An inositol excreting mutant was identified as a phenotypically white colony surrounded by a red halo. The red halo signifies growth of the red indicator strain MC13 in the circular area into which the mutant is excreting inositol.
No quantitative measurement of the amount of inositol excretion by any of the mutants was made.
M. J. White, J. P. Hirsch and S. A. Henry have developed an inositol secreting strain of S. cerevisiae from which the OPI1 gene locus was removed. M. J. Hirsch, S. A. Henry, "The OPT 1 gene of Saccharomyces cerevisiae, a negative regulator of phospholipid biosynthesis, encodes a protein containing polyglutamine tracts and a leucine zipper," J. Biol. Chem., 266, 863-872 (1991). For convenience, this strain is designated the YS2 strain. Removal of the OPI1 locus results in constitutively derepressed expression of inositol-1-phosphate synthase, the product of the INO1 gene. Many of the other enzymes involved in phospholipid biosynthesis are also expressed at high, derepressed levels. Therefore, the OPI1 gene is believed to encode a negative regulator that is required to repress a whole subset of structural genes encoding for phospholipid biosynthetic enzymes. Furthermore, the OPI1 gene was found to be non-essential to the organism since the YS2 strain so constructed is viable and exhibits a phenotype similar to that of previously isolated opi1 mutants. The YS 2 strain accumulates INO1 mRNA constitutively to a level two- to three-fold higher than that observed in the wild-type cells. A sample, designated S. cerevisiae, YS2, was deposited on Feb. 13, 1991 with the American Type Culture Collection located at 12301 Parklawn Drive, Rockville, Md., Accession No. ATCC-74033.
M. J. White and S. A. Henry also developed an inositol-secreting strain of S. cerevisiae from which the OPI1 gene locus was removed and into which multiple copies of the INO1 gene were inserted. Southern blot analysis of the genomic DNA revealed the diploid strain to contain 6 or more copies of the INO1 gene. For convenience, this strain is designated the YS3 strain. A sample, designated S. cerevisiae, YS3, was deposited on Feb. 13, 1991 with the American Type Culture Collection in Rockville, Md., Accession No. ATCC-74034.
Adding the YS2 strain or the YS3 strain to a suitable nutrient medium and adjusting the nutrient medium according to the method of this invention allows significantly enhanced recovery of inositol.